MPRAsnakeflow experiment QC report

TipGood Spearman correlation across replicates!

The data has a good Spearman correlations across replicates using a barcode threshold per oligo of 10! Spearman correlation of DNA is 1.00, RNA is 0.98 and DNA/RNA ratio is 0.96.

Overall quality metrics

Table explanation
  • median rna read count: Median of RNA read count for oligos that passed filtering to determine sufficient coverage in terms of read count. Value is the median of all replicates.
  • median barcodes passing filtering: Median number of barcodes across tested sequences that passed filtering to determine if there was sufficient barcode to oligo coverage. Value is the median of all replicates.
  • pearson correlation: The correlation of log2 RNA/DNA ratios across tested sequences as a measure of replicable activity signal. Value is the median of replicate comparisons using only oligos with >= 10 barcodes.
  • fraction oligos passing: Fraction of tested sequences that passed filtering of the mappable sequences to determine if the designed library was sufficiently recovered. Value is the median of all replicates and using only oligos with >= 10 barcodes.
median barcodes passing filtering median rna read count pearson correlation fraction oligos passing
199 13676 0.97 0.98

DNA over RNA counts

Plotting normalized counts of DNA vs RNA (median across replicates). Only oligos within all replicates are shown. We should see a variation within the RNA count data (along the y axis). If count data between RNA and DNA is highly correlated (e.g. follows the identity line) there is no variation between designed oligos. This is an indication that RNA is inflated with DNA and the DNA digestion before creating cDNA did not work as expected.

Oligo correlation

Oligo correlation plots of DNA, RNA and DNA/RNA ratios across replicates. First tab shows plots using (in average) 2395 oligos with a minimum number of 10 barcodes. Second tab shows all 2438 oligos that have assigned barcodes.

Condition A B #Oligos A #Oligos B #Oligos Joined DNA spearman RNA spearman Ratio spearman DNA log2 pearson RNA log2 pearson Ratio log2 pearson
HEPG2 1 2 2395 2397 2395 1.00 0.98 0.96 1.00 0.98 0.96
HEPG2 1 3 2395 2396 2395 1.00 0.98 0.97 1.00 0.98 0.97
HEPG2 2 3 2397 2396 2396 1.00 1.00 0.99 1.00 1.00 0.99
Condition A B #Oligos A #Oligos B #Oligos Joined DNA spearman RNA spearman Ratio spearman DNA log2 pearson RNA log2 pearson Ratio log2 pearson
HEPG2 1 2 2438 2438 2438 0.99 0.98 0.96 0.99 0.98 0.96
HEPG2 1 3 2438 2438 2438 1.00 0.98 0.97 1.00 0.98 0.97
HEPG2 2 3 2438 2438 2438 1.00 1.00 0.99 0.99 1.00 0.99

Experiment statistic

The total number of oligos in this experiment is 2439 (defined by the assignment) with 1144171 associated barcodes.

In average across replicates we see 2438 from 527485 average barcodes in the count data and around 308720 barcodes where not in the assignment.

condition replicate oligos dna/rna matched barcodes unknown barcodes dna/rna % matched barcodes total dna counts total rna counts avg dna counts per bc avg rna counts per bc barcode outlier removed avg dna/rna barcodes per oligo
HEPG2 1 2438 526817 307782 63.12 21004709 55170350 25.17 66.10 0 216.09
HEPG2 2 2438 523270 227374 69.71 20013767 36061744 26.66 48.04 0 214.63
HEPG2 3 2438 532367 391005 57.65 29614277 55689751 32.07 60.31 0 218.36
Experiment Barcodes Counts Average counts Assigned barcodes Assigned counts Average assigned counts Fraction assigned barcodes Fraction assigned counts
HEPG2.1.DNA 1712563 21963700 12.83 541735 19392468 35.80 0.32 0.88
HEPG2.2.DNA 1643592 20988551 12.77 539266 18561968 34.42 0.33 0.88
HEPG2.3.DNA 2112322 30931193 14.64 551785 27349232 49.57 0.26 0.88
HEPG2.1.RNA 2779664 57511432 20.69 564071 50282968 89.14 0.20 0.87
HEPG2.2.RNA 2199226 37761972 17.17 557727 33004056 59.18 0.25 0.87
HEPG2.3.RNA 2777028 57875580 20.84 564813 50680776 89.73 0.20 0.88

Histograms barcodes per oligo, counts per barcode

Histogramm of number of barcodes per oligo and the number of counts per barcode devidied by DNA and RNA. Median is red, mean is blue.

Activity

Violin and box plots of the log2 fold change of all oligos by the assay. Grouped by labels if set, otherwise NA. First tab shows plots using (in average) 2395 oligos with a minimum number of 10 barcodes. Second tab shows all 2438 oligos that have assigned barcodes.